The establishment and maintenance of spiral ganglion neuron (SGN) connections with hair cells in the cochlea is critical to auditory function, and the disruption of these connections is both a well-recognized consequence of sensorineural hearing loss, and a cause of diminished cochlear implant performance. Despite the importance of these connections, the mechanisms required for SGN axon guidance and synaptogenesis are largely unknown. The long-term goal of this research is to define the mechanisms responsible for auditory nervous system development, so that improved therapies can be developed to treat hearing loss in humans. The results from the research outlined in this application will define how class-3 Semaphorins (Sema3s), a large family of secreted factors that activate Neuropilin/Plexin (Nrp/Plxn) coreceptors on growing axons, are essential for SGN axon guidance decisions. Conceptually, the proposed research is innovative because it will provide the first in-depth analysis of hearing loss caused by SGN axon guidance defects, as there is little known about how the elaboration of SGN fibers within the cochlear duct con-elates with audiological assessments. The proposed research is technically innovative because a mouse model that allows the labeling, visualization and analyses of SGN-hair cell interactions in real time will be used. The function of specific class-3 Semaphorins that are expressed in the cochlea will be defined. Gain-of-function adenovirus infection experiments, and loss-of-function mouse models, will be used to test the hypothesis that Sema3C attracts SGNs and that Sema3A sorts different populations of SGNs. In order to transmit a signal intracellularly, Nrps must bind to Plexin co-receptors. Distinct populations of SGNs differentially express PlexinA3, thus the function of PlexlnA3 will be determined. Using pharmacology, mouse models, and protein truncation experiments, the hypothesis that PlexinA3 activation in different populations of SGNs targets them to specific populations of hair cells will be tested. This contribution of this research will be significant because it will define one of the first molecular signaling pathways to contribute to the development of the complex afferent innervation pattern within the mammalian auditory system.